Spent Culture Media Samples Research Articles

Comparing the advantages, disadvantages and diagnostic power of different non-invasive pre-implantation genetic testing: A literature review.

To improve embryo transfer success and increase the chances of live birth in assisted reproductive methods, there is a growing demand for the use of pre-implantation genetic testing (PGT). However, the invasive approaches used in PGT have led to in vitrofertilization failure and abortions, increasing anxiety levels for parents. To address this, non-invasive PGT methods have been introduced, such as the detection of DNA in blastocoel fluid of blastocysts and spent culture media (SCM). These methods have proven to be minimally invasive and effective in detecting aneuploidy in the chromosomes of human embryos. This review aims to explore the different approaches to pre-implantation diagnosis, including invasive and non-invasive methods, with a particular focus on non-invasive PGT (niPGT). The search strategy involved gathering data from scientific databases such as PubMed, Google Scholar, and Science Direct using relevant keywords. The search was conducted until January 2023. In total, 22 studies have successfully reported the detection and amplification of cell-free DNA in the embryonic SCM. It is important to note that niPGT has some limitations, which include differences in indicators such as cell-free DNA amplification rate, concordance, level of maternal DNA contamination, sensitivity, and specificity between SCM samples and biopsied cells. Therefore, more extensive and detailed research is needed to fully understand niPGT's potential for clinical applications.

A pilot study to investigate the clinically predictive values of copy number variations detected by next-generation sequencing of cell-free deoxyribonucleic acid in spent culture media

To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos. Diagnostic/prognostic accuracy study. Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center. Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed. Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy. One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent. Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A. clinicaltrials.gov (NCT04732013).

P-731 The accuracy of truly non-invasive PGT using spent culture media is insufficient to justify routine clinical use

Abstract Study question Can non-invasive PGT (niPGT), using a protocol that avoids manipulation of the embryo before spent culture medium is collected, accurately determine embryo chromosomal status? Summary answer Accuracy using a truly non-invasive PGT method was low, suggesting this strategy is not appropriate for clinical use and should be restricted to research studies. What is known already PGT requires embryo biopsy to obtain small numbers of cells for genetic testing. The procedure requires skilled personnel and specialist equipment, significantly adding to the cost of PGT while also creating training and logistical bottlenecks that reduce patient access to PGT. Additionally, there have been concerns that biopsy could damage embryos. These considerations have stimulated interest in potential non-invasive PGT strategies. Thus far, niPGT methods have mostly focused on cell-free embryonic DNA found in spent culture media, but studies reporting highest accuracy have tended to involve embryo manipulations likely to promote DNA release and therefore cannot be considered truly non-invasive. Study design, size, duration Samples of spent culture medium (SCM) associated with 128 embryos were collected. Oocytes had been denuded of cumulus cells, fertilised using ICSI and the resulting embryos thoroughly washed on day-4 before transfer to a fresh drop of medium. SCM samples were collected on day-5 or day-6 of culture. Embryos had not previously been cryopreserved and underwent minimal manipulation prior to SCM collection. Results of SCM analysis were compared to those obtained following conventional ‘invasive’ PGT. Participants/materials, setting, methods Media samples were subjected to whole genome amplification using the PG-Seq Rapid Non-Invasive PGT kit. Concentrations of sequencing libraries were measured, and next-generation sequencing (NGS) was undertaken. The resulting NGS data was analysed with PG-Find Software to predict chromosomal status. In parallel, trophectoderm biopsies from the corresponding embryos were analysed using a well-established and highly validated PGT method, based upon amplification of thousands of sites across the genome, followed by NGS. Main results and the role of chance Despite the implementation of rigorous measures to prevent contamination of SCM samples, extraneous female DNA, likely of cumulus cell origin, was frequently observed. Depending on the clinic, 16.7% to 38.1% of media samples associated with male embryos gave a discordant (female) result. This highlights the difficulty of avoiding inadvertent sampling of maternal DNA. There were no instances of male DNA contamination affecting SCM samples from female embryos. We assessed whether clinic of origin, ploidy status of the embryo, and day of SCM collection influenced the quality of the sequencing libraries (ANOVA). Higher concentrations of libraries and superior sequencing quality scores were observed in SCM collected on day-6 compared to day-5 (p = 0.011 and p = 0.002, respectively). Chromosomal assessments obtained from SCM were compared to those from standard, trophectoderm-based PGT in a blinded fashion. 50% of SCM samples gave an identical predicted karyotype to their corresponding trophectoderm biopsy (concordant copy number for all chromosomes). When applying a simple aneuploid/euploid classification, 80% of embryos classified chromosomally abnormal using the validated PGT method were also aneuploid according to analysis of the associated SCM sample, but without necessarily having identical karyotypes, while 62% of embryos classified euploid by PGT received the same designation after SCM analysis. Limitations, reasons for caution SCM samples were designated ‘contaminated’ when embryos of one sex (determined using invasive PGT) were incorrectly classified as the opposite sex using niPGT. However, our study was unable to determine when SCM samples were contaminated with DNA of the same sex as the embryo. Consequently, contamination rates could be higher. Wider implications of the findings Most niPGT methods are not truly non-invasive, as they include embryo manipulations that increase likelihood of cell death and DNA release. Our results show that obtaining reliable niPGT results is difficult without deviating significantly from optimal embryological protocols. Currently, the accuracy of niPGT seems insufficient to justify routine clinical use. Trial registration number Not applicable

P-563 The effect of embryo culture time on concordance rates between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent culture media (SCM) analysis

Abstract Study question To evaluate different embryo culture times (day5 versus day6) to understand the ideal time-frame for the performance of concordance analysis between invasive and non-invasive PGT-A(niPGT-A) Summary answer Different levels of ploidy concordance rates were observed between day5 and day6 groups: 72,6% versus 84,8%, respectively What is known already The recent data supports that a non-invasive approach for evaluating embryo ploidy status may be an alternative to standard invasive methods. Embryo cell-free DNA(cfDNA) released into culture media during in-vitro embryo development represents the potential source for this analysis. The release of cfDNA from embryos is expected to be directly proportional to embryo culture time and late-stage released cfDNA may be more representative of the embryo. Therefore, it is important to estimate the most effective time frame of the culture that will provide the most conclusive data from spent-culture-media(SCM) without adversely affecting the development of the embryo Study design, size, duration A total of 334 SCM from blastocyst stage embryos have been included in this study. The SCM samples were divided in 2 subgroups according to the embryo culture time as day5 (n = 154) and day6 (n = 180). The cfDNA of SCM samples were amplified by whole genome amplification(WGA) and analyzed by next generation sequencing(NGS) in parallel to day5 or day6 trophectodermal(TE) biopsied samples of their corresponding embryos. Ploidy status and concordance were compared between two groups Participants/materials, setting, methods Day3 embryos were washed and transferred in 20µl fresh culture media until the biopsy. SCM samples were collected in PCR tubes and stored at -20 °C while embryos were biopsied for standard PGT-A analysis. Both SCM and corresponding TE biopsy samples were amplified by Sureplex(Vitrolife). Then TE and SCM samples were analyzed by next-generation sequencing(NGS) using MiSeq® System (Illumina). Data analysis has been done by Bluefuse Multi Software 4.5(Illumina) for all SCM and TE samples Main results and the role of chance A successful DNA amplification rate (>30ng/µl) was obtained in both day5 and day6 groups with 95,5% (147/154) and 99,4% (179/180) respectively. The DNA concentration after WGA was ranging between 30.1-122.5ng/µl and 38.5-123.2ng/µl in day5 and day6 groups respectively. Not conclusive (NC) results including a noisy NGS profile and chaotic chromosome aneuploidies (>5 chromosomes) were excluded in both groups. Therefore, 64,6% (95/147) of SCM samples in day5 group and 81,0% (145/179) of SCM samples in day6 group were conclusive for NGS analysis. The ploidy concordance rate between SCM and TE samples (euploid vs euploid, aneuploid vs aneuploid) was 72,6% (69/95) in day5 and 84,8% (123/145) in day6 group. In day5 group, the false-negative rate was 10.5% (10/95), and false-positive rate was 16.8% (16/95) while in day6 group the false-negative rate was 6,8% (10/145), and false-positive rate was 8,2% (12/145). Sensitivity and specificity were calculated as 83,6% and 52,9% in day5 group, and 90,9% and 65,7 in day6 group respectively Limitations, reasons for caution We have high number of samples in our study, but larger prospective studies may change the significance of ploidy concordance. One of the important issues in SCM analysis is the maternal DNA contamination risk which cannot be revealed always. Therefore the use of molecular markers would increase the reliability Wider implications of the findings A non-invasive approach for aneuploidy screening may be an alternative to standard PGT-A procedure with invasive embryo biopsy but the evaluation of ideal culture time is crucial for the performance of niPGT-A method. Our study demonstrates that the late collection time of SCM provides significantly better ploidy concordance rates Trial registration number Not applicable

Clinical Relevance of Secreted Small Noncoding RNAs in an Embryo Implantation Potential Prediction at Morula and Blastocyst Development Stages.

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30–40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers.

NON-INVASIVE PREIMPLANTATION GENETIC TESTING (NIPGT) FOR ANEUPLOIDY AND STRUCTURAL REARRANGEMENTS USING NEXT GENERATION SEQUENCING (NGS) ON SPENT CULTURE MEDIA (SCM)

Non-invasive preimplantation genetic testing (niPGT) for aneuploidy (A) and structural rearrangements (SR) assesses ploidy indirectly by analyzing embryonic cell-free DNA in the spent culture media (SCM). In this study we investigated the predictive values of niPGTA/SR by analyzing the SCM samples of embryos, previously analyzed by trophectoderm (TE) biopsy, to compare ploidy concordance rates between TE, SCM and whole embryos.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (>84%) of concordance rate for ploidy and sex, sensitivity (>88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (>5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

Validation of preimplantation genetic tests for aneuploidy (PGT-A) with DNA from spent culture media (SCM): concordance assessment and implication

BackgroundSpent culture medium (SCM) as a source of DNA for preimplantation genetic tests aneuploidy (PGT-A) has been widely discussed.MethodsSeventy-five blastocysts that were donated for research provided a unique possibility in which multiple specimens, including trophectoderm (TE) biopsy, SCM, and paired corresponding whole blastocyst (WB) specimens from the same blastocyst source, could be utilized for the purpose of this preclinical validation.ResultsTo conduct a validation ploidy concordance assessment, we evaluated the full chromosomal concordance rates between SCM and WB (SCM-to-WB), and between TE and WB (TE-to-WB) as well as sensitivity, specificity and overall diagnostic accuracy. 78.67% (59/75) of NGS results in the SCM group were interpretable, a significantly lower percentage than their corresponding TE and WB groups. This discrepancy manifests itself in intrinsically low quantity and poor integrity DNA from SCM. Subsequently, remarkable differences in full concordance rates (including mosaicism, and segmental aneuploidies) are seen as follows: 32.2% (SCM-to-WB, 19/59) and 69.33% (TE-to-WB, 52/75), (p < 0.001). In such cases, full concordance rates were 27.27% (15/55) in SCM-to-WB, and, 76% (57/75) in TE-to-WB (p < 0.001). Collectively, the NGS data from SCM also translated into lower sensitivities, Positive Predictive Value (PPV), Negative Predictive Value (NPV), overall diagnostic accuracies, and higher Negative Likelihood Ratio (NLR).ConclusionsOur study reveals that DNA is detectable in the majority of SCM samples. Individual chromosomal aberration, such as segmental aneuploidy and mosaicism, can be quantitatively and qualitatively measured. However, TE still provides a more accurate and reliable high-throughput methodology for PGT-A. Meanwhile, cell-free DNA in SCM reporting lacks uniform diagnostic interpretations. Considering that this test is meant to determine which embryos are relegated to be discarded, PGT-A with cell-free DNA in SCM should not be permitted to be applied in routine clinical settings for diagnosis purpose.

Apoptosis triggers the release of microRNA miR-294 in spent culture media of blastocysts

PurposeTo study whether members of the miR-290-295 cluster in spent culture medium (SCM) of embryos are correlated with morphokinetics and apoptosis.MethodsCryopreserved 1-cell stage mouse embryos were cultured to the blastocyst stage, development was monitored by time-lapse, 59 SCM were collected, and miR-291a and miR-294 were detected with polymerase chain reaction (PCR). Blastocysts were immuno-stained for sexing (H2AK119ub) and for apoptosis (TUNEL). Each embryo and SCM were individually processed. Correlations were run between the miRNAs and developmental events (t2, t3, t4, t5, t8, tSB, tB, ECC2, ECC3, s2, s3, dB) and apoptosis (apoptotic cells/total cell number %). MiR-294 SCM and cell levels were compared in 40 blastocysts. Apoptosis was induced in 15 blastocysts with UV radiation and SCM samples were analyzed for miR-294.ResultsMiR-291a and miR-294 are released in variable levels by mouse blastocysts. Their release is similar between male and female embryos. No significant correlations were found between these miRNAs and development. MiR-294 was significantly positively correlated with apoptosis (r = 0.560, p < 0.001). Cellular expression was lower in blastocysts that released miR-294 in high levels compared with null, low, and medium release embryos (p < 0.01). UV radiation caused apoptosis which triggered higher secretion of miR-294 in 15 blastocysts versus 13 control embryos (p < 0.01).Conclusion(s)MicroRNAs are important regulators of preimplantation development. Apoptosis triggers the release of miR-294 by blastocysts which possibly serves a secretory role for embryo-maternal communication. SCM miRNA analysis is possible for individually cultured embryos and future studies can investigate miRNAs as noninvasive markers of embryo quality.

MicroRNAs in combined spent culture media and sperm are associated with embryo quality and pregnancy outcome

To identify differentially abundant miRNAs in sperm samples and spent culture media (SCM) of embryos of different grade toward a prediction of pregnancy outcome. Array-based reverse-transcription quantitative polymerase chain reaction profiling and validation. University research institute and invitro fertilization center. Couples (n = 61) undergoing infertility treatment with the use of intracytoplasmic sperm injection. None. Abundance levels of miRNAs in combined SCM of embryos of different quality and in sperm samples associated with pregnancy outcome. Out of 372 screened miRNAs, miR-19b-3p and let-7a-5p were detected consistently in all SCM and sperm samples. The abundance levels of miRNAs were significantly altered between SCM of embryos with different quality (G1, G2, and G3 grades). Specifically, miR-320a and miR-15a-5p were differentially abundant in G1 vs. G2, miR-21-5p in G1 vs. G3, and miR-20a-5p in G2 vs. G3. The abundance levels of combined SCM and sperm derived miRNAs were also significantly altered between different pregnancy outcomes. MiR-19b-3p showed the highest area under the receiver operating characteristic curve values between positive and negative outcomes, with lower abundance levels in both combined SCM and sperm samples associated with a positive pregnancy outcome. MiR-320a, miR-15a-5p, miR-21-5p, and miR-20a-5p showed similar results in combined SCM samples. miRNA abundance levels in combined SCM and sperm differed significantly depending on embryo quality and pregnancy outcome. MiR-19b-3p may serve as a potential biomarker to predict pregnancy outcome.

Ozone oxidation pretreatment for enzymatic saccharification of spent culture media after Lentinula edodes cultivation

Ozone oxidation pretreatment was carried out on spent culture media (SCMs) after Lentinula edodes cultivation to improve fermentable sugar production by enzymatic saccharification. SCM samples treated with ozone for different treatment time under various moisture contents (MCs) were enzymatically saccharified, and hydrolysis weight decrease, reducing sugar yield, and monosaccharide yields were determined. Klason lignin content in SCM samples with 20 and 40 % MC was greatly decreased by ozone pretreatment for all ozone treatment time. In addition, in the ozone-treated sample at 40 % MC, the highest hydrolysis weight decrease after saccharification was obtained in the SCM sample treated for 1 h. After saccharification, glucose yield from the ozone-treated sample for 1 h was higher than that treated for 2 h. These results indicate that ozone treatment is effective to improve fermentable sugar production by enzymatic saccharification for the SCMs of L. edodes.